The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has become an extremely useful tool for tracking and quantifying biological entities in the fields of biochemistry, molecular and cell biology, and medical diagnostics (Chalfie et al., 1994, Science 263: 802-805; Tsien, 1998, Ann, Rev. Biochem. 67: 509-544). There are no cofactors or substrates required for fluorescence, thus the protein can be used in a wide variety of organisms and cell types. GFP has been used as a reporter gene to study gene expression in vivo by insertion downstream of a test promoter. The protein has also been used to study the subcellular localization of a number of proteins by direct fusion of the test protein to GFP, and GFP has become the reporter of choice for monitoring the infection efficiency of viral vectors both in cell culture and in animals. In addition, a number of genetic modifications have been made to GFP resulting in variants for which spectral shifts correspond to changes in the cellular environment such as pH, ion flux, and the phosphorylation state of the cell. Perhaps the most promising role for GFP as a cellular indicator is its application to fluorescence resonance energy transfer (FRET) technology. FRET occurs with fluorophores for which the emission spectrum of one overlaps with the excitation spectrum of the second. When the fluorophores are brought into close proximity, excitation of the xe2x80x9cdonorxe2x80x9d fluorophore results in emission from the xe2x80x9cacceptorxe2x80x9d. Pairs of such fluorophores are thus useful for monitoring molecular interactions. Fluorescent proteins such as GFP are useful for analysis of protein:protein interactions in vivo or in vitro if their fluorescent emission and excitation spectra overlap to allow FRET. The donor and acceptor fluorescent proteins may be produced as fusions with the proteins one wishes to analyze for interactions. These types of applications of GFPs are particularly appealing for high throughput analyses, since the readout is direct and independent of subcellular localization.
Purified A. victoria GFP is a monomeric protein of about 27 kDa that absorbs blue light with excitation wavelength maximum of 395 nm, with a minor peak at 470 nm, and emits green fluorescence with an emission wavelength of about 510 nm and a minor peak near 540 nm (Ward et al., 1979, Photochem. Photobiol. Rev. 4: 1-57). The excitation maximum of A. victoria GFP is not within the range of wavelengths of standard fluorescein detection optics. Further, the breadth of the excitation and emission spectra of the A. victoria GFP are not well suited for use in applications involving FRET. In order to be useful in FRET applications, the excitation and emission spectra of the fluorophores are preferably tall and narrow, rather than low and broad. There is a need in the art for GFP proteins that are amenable to the use of standard fluorescein excitation and detection optics. There is also a need in the art for GFP proteins with narrow, preferably non-overlapping spectral peaks.
The use of A. victoria GFP as a reporter for gene expression studies, while very popular, is hindered by relatively low quantum yield (the brightness of a fluorophore is determined as the product of the extinction coefficient and the fluorescence quantum yield). Generally, the A. victoria GFP coding sequences must be linked to a strong promoter, such as the CMV promoter or strong exogenous regulators such as the tetracycline transactivator system, in order to produce readily detectable signal. This makes it difficult to use GFP as a reporter for examining the activity of native promoters responsive to endogenous regulators. Higher intensity would obviously also increase the sensitivity of other applications of GFP technology. There is a need in the art for GFP proteins with higher quantum yield.
Another disadvantage of A. victoria GFP involves fluctuations in its spectral characteristics with changes in pH. At high pH (pH 11-12), the wild-type A. victoria GFP loses absorbance and excitation amplitude at 395 nm and gains amplitude at 470 nm (Ward et al., 1982, Photochem. Photobiol. 35: 803-808). A. victoria fluorescence is also quenched at acid pH, with a pKa around 4.5. There is a need in the art for GFPs exhibiting fluorescence that is less sensitive to pH fluctuations.
Further, in order to be more useful in a broad range of applications, there is a need in the art for GFP proteins exhibiting increased stability of fluorescence characteristics relative to A. victoria GFP, with regard to organic solvents, detergents and proteases often used in biological studies. There is also a need in the art for GFP proteins that are more likely to be soluble in a wider range of cell types and less likely to interfere non-specifically with endogenous proteins than A. victoria GFP.
A number of modifications to A. victoria GFP have been made with the aim of enhancing the usefulness of the protein. For example, modifications aimed at enhancing the brightness of the fluorescence emissions or the spectral characteristics of either the excitation or emission spectra or both have been made. It is noted that the stated aim of several of these modification approaches was to make an A. victoria GFP that is more similar to R. reniformis GFP in its excitation and emission spectra and fluorescence intensity.
Literature references relating to A. victoria mutants exhibiting altered fluorescence characteristics include, for example, the following. Heim et al. (1995, Nature 373: 663-664) relates to mutations at S65 of A. victoria that enhance fluorescence intensity of the polypeptide. The S65T mutation to the A. victoria GFP is said to xe2x80x9cameliorate its main problems and bring its spectra much closer to that of Renillaxe2x80x9d.
A review by Chalfie (1995, Photochem. Photobiol. 62: 651-656) notes that an S65T mutant of A. victoria, the most intensely fluorescent mutant of A. victoria known at the time, is not as intense as the R. reniformis GFP.
Further references relating to A. victoria mutants include, for example, Ehrig et al., 1995, FEBS Lett. 367: 163-166); Surpin et al., 1987, Photochem. Photobiol. 45 (Suppl): 95S; Delagrave et al., 1995, BioTechnology 13: 151-154; and Yang et al., 1996, Gene 173: 19-23.
Patent and patent application references relating to A. victoria GFP and mutants thereof include the following. U.S. Pat. No. 5,874,304 discloses A. victoria GFP mutants said to alter spectral characteristics and fluorescence intensity of the polypeptide. U.S. Pat. No. 5,968,738 discloses A. victoria GFP mutants said to have altered spectral characteristics. One mutation, V163A, is said to result in increased fluorescence intensity. U.S. Pat. No. 5,804,387 discloses A. victoria mutants said to have increased fluorescence intensity, particularly in response to excitation with 488 nm laser light. U.S. Pat. No. 5,625,048 discloses A. victoria mutants said to have altered spectral characteristics as well as several mutants said to have increased fluorescence intensity. Related U.S. Pat. No. 5,777,079 discloses further combinations of mutations said to provide A. victoria GFP polypeptides with increased fluorescence intensity. International Patent Application (PCT) No. W098/21355 discloses A. victoria GFP mutants said to have increased fluorescence intensity, as do W097/20078, W097/42320 and W097/11094. PCT Application No. W098/06737 discloses mutants said to have altered spectral characteristics, several of which are said to have increased fluorescence intensity.
In addition to A. victoria, GFPs have been identified in a variety of other coelenterates and anthazoa, however only three GFPs have been cloned, those from A. victoria (Prasher, 1992, Gene 111: 229-233) and from the sea pansies, Renilla mulleri (WO 99/49019) and Renilla reniformis (Felts et al. (2000) Strategies 13:85). One common drawback that all three of the cloned GFPs share is relatively poor expression in mammalian cells.
The present invention provides a humanized polynucleotide encoding R. mulleri GFP.
In a preferred embodiment, the polynucleotide comprises the sequence of SEQ ID NO: 1.
In one embodiment, the invention provides a recombinant vector comprising a humanized polynucleotide encoding R. mulleri GFP.
In a further embodiment, the recombinant vector is contained within a cell.
The present invention further provides a method of producing R. mulleri GFP comprising the steps of: introducing a recombinant vector comprising a humanized polynucleotide sequence encoding R. mulleri GFP to a cell; culturing the cell; and isolating R. mulleri GFP from the cell.
In one embodiment, the cell is a mammalian cell.
In a preferred embodiment, the cell is a human cell.
The present invention further provides a method of determining the location of a polypeptide of interest in a cell, the method comprising the steps of: linking said polynucleotide sequence encoding a polypeptide of interest with a humanized polynucleotide encoding R. mulleri GFP, such that the linked polynucleotide sequences are fused in frame; introducing the linked polynucleotide sequences to a cell; and determining the location of the polypeptide encoded by the linked polynucleotide sequences.
The invention also provides a method of identifying cells to which a recombinant vector has been introduced, the method comprising the steps of: introducing a recombinant vector to a population of cells, wherein the recombinant vector comprises a humanized polynucleotide which encodes R. mulleri GFP and the cells permit expression of said humanized polynucleotide; illuminating the cell population with light within the excitation spectrum of R. mulleri GFP; and detecting fluorescence in the emission spectrum of R. mulleri GFP in the cell population, thereby identifying a cell to which said recombinant vector has been introduced.
In one embodiment, the GFP is expressed as a fusion polypeptide.
In a further embodiment, the GFP is expressed as a distinct polypeptide.
In one embodiment, the cells are identified by FACS analysis.
The invention further provides a method of monitoring the activity of a transcriptional regulatory sequence, the method comprising the steps of: operably linking a nucleic acid sequence comprising the transcriptional regulatory sequence to a humanized nucleic acid sequence encoding R. mulleri GFP to form a reporter construct; introducing the reporter construct to a cell; and detecting R. mulleri GFP fluorescence in the cell, wherein the fluorescence reflects the activity of the transcriptional regulatory sequence.
The invention still further provides a method of detecting a modulator of a transcriptional regulatory sequence, the method comprising the steps of: operably linking a nucleic acid sequence comprising the transcriptional regulatory sequence to a humanized nucleic acid sequence encoding R. mulleri GFP to form a reporter construct, wherein the transcriptional regulatory sequence is responsive to the presence of the modulator; introducing the reporter construct to a cell; and detecting R. mulleri GFP fluorescence in the cell, wherein the fluorescence indicates the presence of the modulator.
The invention still further provides a method of screening for an inhibitor of a transcriptional regulatory sequence, the method comprising the steps of: operably linking a nucleic acid sequence comprising the transcriptional regulatory sequence to a humanized nucleic acid sequence encoding R. mulleri GFP to form a reporter construct; introducing the reporter construct to a cell; contacting the cell with a candidate inhibitor of the transcriptional regulatory sequence; and detecting R. mulleri GFP fluorescence in the cell, wherein a decrease in the fluorescence relative to that detected in the absence of the candidate inhibitor indicates that the candidate inhibitor inhibits the activity of the transcriptional regulatory sequence.
The invention still further provides a method of producing a fluorescent molecular weight marker, the method comprising the steps of: linking a humanized nucleic acid sequence encoding R. mulleri GFP in frame to a nucleic acid sequence encoding a polypeptide of known relative molecular weight such that the linked molecules encode a fusion polypeptide; introducing the linked nucleic acid sequences to a cell; isolating said fusion polypeptide from the cell, wherein the fusion polypeptide is a relative molecular weight marker.
In one embodiment, the cell is a mammalian cell.
In a further embodiment, the cell is a human cell.
In a still further embodiment, the humanized nucleic acid sequence encoding R. mulleri GFP is the sequence of SEQ ID NO: 1.
The term xe2x80x9chumanized R. mulleri polynucleotidexe2x80x9d or xe2x80x9chumanized R. mulleri GFP sequencexe2x80x9d refers to a polynucleotide coding sequence in which at least 179 codons of the polynucleotide coding sequence for a non-human polypeptide (i.e., a polypeptide not naturally expressed in humans) have been altered to a codon sequence more preferred for expression in mammalian cells (i.e., SEQ ID NO: 1). In the xe2x80x9chumanized R. mulleri GFP nucleotide sequence of SEQ ID NO: 1, residue number 93 may be either a T or a C. In addition, an equivalent of a humanized sequence according to the invention is contempalted which is a polynucleotide according to SEQ ID NO: 1 in which one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, or twenty of those 179 codons that are altered to be humanized codons in SEQ ID NO: 1 are not altered such that they are humanized codons (that is, are not preferred in mammalian, particularly human, cells), provided expression in mammalian cells of the equivalent xe2x80x9chumanized R. mulleri polynucleotidexe2x80x9d described in SEQ ID NO: 1 is not reduced (relative to expression of the humanized sequence of SEQ ID NO: 1 in the same type of cells) by more than 5% or at most 10%.
The amount of fluorescent polypeptide expressed in a human cell from a humanized GFP polynucleotide sequence is at least two-fold greater, on either a mass or a fluorescence intensity scale per cell, than the amount expressed from an equal amount or number of copies of a wild type R. mulleri GFP polynucleotide.
As used herein, the term xe2x80x9chumanized codonxe2x80x9d means a codon, within a polynucleotide sequence encoding a non-human polypeptide, that has been changed to a codon that is more preferred for expression in human cells relative to that codon encoded by the non-human organism from which the non-human polypeptide is derived. Species-specific codon preferences stem in part from differences in the expression of tRNA molecules with the appropriate anticodon sequence. That is, one factor in the species-specific codon preference is the relationship between a codon and the amount of corresponding anticodon tRNA expressed.
It should be understood that any of the recombinant vectors of the invention or cells containing such a vector will comprise a humanized polynucleotide encoding R. mulleri GFP.
The wild type xe2x80x9cR. mulleri green fluorescent proteinxe2x80x9d or xe2x80x9cR. mulleri GFPxe2x80x9d is encoded by the nucleic acid sequence of SEQ ID NO: 2 (WO 99/49019, incorporated herein by reference).
As used herein, the term xe2x80x9cwild-type R. mulleri GFPxe2x80x9d refers to a polypeptide of SEQ ID NO: 3 (WO 99/49019).
The term xe2x80x9cvariant thereofxe2x80x9d when used in reference to an R. mulleri GFP means that the amino acid sequence bears one or more residue differences relative to the wild type R. mulleri GFP sequence and has the identical biological activity (fluorescence intensity) of the wild type polypeptide.
As used herein, the term xe2x80x9cincreased fluorescence intensityxe2x80x9d or xe2x80x9cincreased brightnessxe2x80x9d refers to fluorescence intensity or brightness that is greater than that exhibited by wild-type R. mulleri GFP under a given set of conditions. Generally, an increase in fluorescence intensity or brightness means that fluorescence of a variant is at least 5% or more, and preferably 10%, 20%, 50%, 75%, 100% or more, up to even 5 times, 10 times, 20 times, 50 times or 100 times or more intense or bright than wild-type R. mulleri GFP under a given set of conditions.
As used herein, the term xe2x80x9cfused heterologous polypeptide domainxe2x80x9d refers to an amino acid sequence of two or more amino acids fused in frame to R. mulleri GFP. A fused heterologous domain may be linked to the N or C terminus of the R. mulleri GFP polypeptide.
As used herein, the term xe2x80x9cfused to the amino-terminal endxe2x80x9d refers to the linkage of a polypeptide sequence to the amino terminus of another polypeptide. The linkage may be direct or may be mediated by a short (e.g., about 2-20 amino acids) linker peptide.
As used herein, the term xe2x80x9cfused to the carboxy-terminal endxe2x80x9d refers to the linkage of a polypeptide sequence to the carboxyl terminus of another polypeptide. The linkage may be direct or may be mediated by a linker peptide.
As used herein, the term xe2x80x9clinker sequencexe2x80x9d refers to a short (e.g., about 1-20 amino acids) sequence of amino acids that is not part of the sequence of either of two polypeptides being joined. A linker sequence is attached on its amino-terminal end to one polypeptide or polypeptide domain and on its carboxyl-terminal end to another polypeptide or polypeptide domain.
As used herein, the term xe2x80x9cexcitation spectrumxe2x80x9d refers to the wavelength or wavelengths of light that, when absorbed by a fluorescent polypeptide molecule of the invention, causes fluorescent emission by that molecule.
As used herein, the term xe2x80x9cemission spectrumxe2x80x9d refers to the wavelength or wavelengths of light emitted by a fluorescent polypeptide.
As used herein, the terms xe2x80x9cdistinguishablexe2x80x9d or xe2x80x9cdetectably distinctxe2x80x9d mean that standard filter sets allow either the excitation of one form of a polypeptide without excitation of another given polypeptide, or similarly, that standard filter sets allow the distinction of the emission from one polypeptide form from the emission spectrum of another. Generally, distinguishable or detectably distinct excitation or emission spectra have peaks that vary by more than 1 nm, and preferably vary by more than 2, 3, 4, 5, 10 or more nm.
As used herein, the term xe2x80x9cfusion polypeptidexe2x80x9d refers to a polypeptide that is comprised of two or more amino acid sequences, from two or more proteins that are not found linked in nature, that are physically linked by a peptide bond. As used herein, only one protein which comprises a xe2x80x9cfusion polypeptidexe2x80x9d of the present invention is a fluorescent protein.
As used herein, the term xe2x80x9cemission spectrum overlaps the excitation spectrumxe2x80x9d means that light emitted by one fluorescent polypeptide is of a wavelength or wavelengths that causes excitation and emission by another fluorescent polypeptide.
As used herein, the term xe2x80x9cpopulation of cellsxe2x80x9d refers to a plurality of cells, preferably, but not necessarily of same type or strain.
As used herein the term xe2x80x9cdistinct polypeptidexe2x80x9d refers to a polypeptide that is not expressed as a fusion polypeptide.
As used herein, the term xe2x80x9cFACS analysis xe2x80x9d refers to the method of sorting cells, fluorescence activated cell sorting, wherein cells are stained with or express one or more fluorescent markers. In this method, cells are passed through an apparatus that excites and detects fluorescence from the marker(s). Upon detection of fluorescence in a given portion of the spectrum by a cell, the FACS apparatus allows the separation of that cell from those not expressing that fluorescence spectrum.
As used herein, the term xe2x80x9clipid soluble transcriptional modulatorxe2x80x9d refers to a composition that is capable of passing through cell membranes (nuclear or cytoplasmic) and has a positive or negative effect on the transcription of one or more genes or constructs.
As used herein, the term xe2x80x9coperably linkedxe2x80x9d means that a given coding sequence is joined to a given transcriptional regulatory sequence such that transcription of the coding sequence occurs and is regulated by the regulatory sequence.
As used herein, the term xe2x80x9creporter constructxe2x80x9d refers to a polynuclectide construct encoding a detectable molecule, linked to a transcriptional regulatory sequence conferring regulated transcription upon the polynucleotide encoding the detectable molecule. A detectable molecule is preferably an R. mulleri GFP.
As used herein, the term xe2x80x9cresponsive to the presence of a modulatorxe2x80x9d means that a given transcriptional regulatory sequence is either turned on or turned off in the presence of a given compound. As used herein, gene expression is xe2x80x9cturned onxe2x80x9d when the polypeptide encoded by the gene sequence (e.g., a GFP polypeptide) is detectable over background, or alternatively, when the polypeptide is detectable in an increased amount over the amount detected in the absence of a given modulator compound. In this context, xe2x80x9cincreased amountxe2x80x9d means at least 10%, preferably 20%, 50%, 75%, 100% or more, up to even 5 times, 10 times, 20 times, 50 times, or 100 times or more higher than background detection, with background detection being the amount of signal observed in the absence of the modulator compound.
As used herein, the term xe2x80x9cmodulator of a transcriptional regulatory sequencexe2x80x9d refers to a compound or chemical moiety that causes a change in the level of expression from a transcriptional regulatory sequence. Preferably, the change is detectable as an increase or decrease in the detection of a reporter molecule or reporter molecule activity, with at least 10%, 20%, 50%, 75%, 100%, or even 5 times, 10 times, 20 times, 50 times or 100 times or more increased or decreased level of reporter signal relative to the absence of a given modulator.
As used herein the term xe2x80x9cinhibitor of a transcriptional regulatory sequencexe2x80x9d refers to a compound or chemical moiety that causes a decrease in the amount of a reporter molecule or reporter molecule activity expressed from a given transcriptional regulatory sequence. As used herein, the term xe2x80x9cdecreasexe2x80x9d when used in reference to the detection of a reporter molecule or reporter molecule activity means that detectable activity is reduced by at least 10%, 20%, 50%, 75%, or even 100% (i.e., no expression), relative to the amount detected in the absence of a given compound or chemical moiety. As used herein the term xe2x80x9ccandidate inhibitorxe2x80x9d refers to a compound or chemical moiety being tested for inhibitory activity in an assay.
An advantage of the present invention is that it provides a method for the improved expression of a GFP in mammalian, particularly human cells both in vivo and in vitro. A further advantage of the present invention is that it provides a method of providing a humanized R. Mulleri GFP which, due to enhanced expression will produce a stronger fluorescent signal in cells in which it is expressed.
Further features and advantages of the invention will become more fully apparent in the following description of the embodiments and drawings thereof, and from the claims.